Gene mutation refers to the structure and function change of a genome DNA molecule in compositions or sequence of the base pairs, mainly including base substitution and the deletion or insertion of a small fragment, which is one of the major causes for the genetic diseases. Polymorphism refers to accumulated changes of DNA sequence in human beings during evolution. Gene mutation and polymorphism analysis play very important roles in biomedical researches, particularly in diagnosis and pathological study of genetic diseases. With the development of the human whole-genome sequencing project, it becomes a very urgent task to study gene mutation and polymorphism.
Gene mutations can be determined in many ways. The most classical gene mutation detection technique is nucleic acid hybridization. The traditional methods of nucleic acid molecular hybridization include blot hybridization on a membrane (such as Southern blot, Northern blot), cell hybridization in situ, etc. Due to its high hybridization specificity and high detection sensitivity, the application of nucleic acid hybridization has significantly promoted the development of molecular biology. However, traditional nucleic acid hybridization has a complicated process and too many operation steps, and especially the probes used in the methods are often radioactively labeled, which may easily damage human bodies. Therefore, there is an urgent need for novel, fast and safe detection and analysis technique.
Since PCR technology was developed in 1985, gene mutation detection techniques have been developed rapidly, and many novel detection means and technologies have derived from them, for PCR technology has a powerful in vitro amplification ability for DNA, and the sensitivity and specificity of PCR are greatly enhanced when it is used in combination with a nucleic acid hybridization technology. Many of those techniques are suitable for detecting point mutations, as well as SNPs. Currently, the most popular PCR-based detection technologies include: the methods for detection of known mutations and SNPs include: allele-specific oligonucleotide hybridization (ASO), ligase chain reaction (LCR), TaqMan technology, polymerase chain reaction-based restriction fragment length polymorphism analysis (PCR-RFLP), short tandem repeat length polymorphism (STR), etc.; and the methods for detection of unknown mutations includes: single-strand conformation polymorphism (SSCP), heteroduplex polymorphism analysis (HPA), MALDI-TOF mass spectrometry (matrix assisted laser desorptionion Ization time of flight mass spectrometry), denaturing gradient gel electrophoresis (DGGE), enzyme mismatch cleavage (EMC), dideoxy fingerprinting (ddF), DNA sequencing, etc. The characteristics, principles and applications of those methods have been described in many previous publications. Although these methods can be used to detect the presence of a mutation, most of the methods cannot identify the type of the mutation and can only detect a part of SNP; meanwhile, most of the methods, such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, high performance liquid chromatography and the like, require detection means such as post PCR processing to analyze and identify the results, and the identification of results is quite complicated. In addition, most of the above detection technologies have complicated processing steps, and only a few of the samples can be detected once, so it is difficult to meet the needs of automation.
DNA sequencing is the most fundamental method for mutation detection. Although DNA sequencing can accurately identify the location and type of a mutation, its practical application is limited for the current gel electrophoresis-based sequencing technology is time-consuming, while the sequencing with automated sequencer is costly.
Biological microarray (or biochip) technology is a fast and high-throughput detection tool that was developed in recent years. In the biological microarray technology, which takes advantage of microarray technology, thousands of biological components (cells, proteins, DNA, etc.) are arranged on a solid phase substrate. The components being detected in a biological sample react with a specific substance on the substrate, and then an appropriate signal (such as fluorescence) is introduced to achieve the purpose of analysis of the biological sample. Biological microarray technology makes some traditional biological analysis means to proceed in a space as small as possible and in a rate as fast as possible. At present, the biochip technology are developing rapidly, and researchers have used a variety of chip technologies, such as gene chip, protein chip, tissue chip, cell chip, lab-on-chip and the like, for large-scale mutation detection and polymorphism screen.
Currently, there are a variety of methods and patent technologies that are based on a DNA chip for gene mutation detections (such as SNP detection) and nucleic acid sequencing analysis, such as Beckman's SNP throughput analysis System (SNPStream assay (Orchid Cellmark/Beckman Coulter)), Illumina's GoldenGate multiple site-specific extension amplification classification system (GoldenGate genotyping assay (Illumina)), Affymetrix's human whole genomic mapping analysis system (GeneChip Human Mapping assays (Affymetrix)), Illumina's Infinium genotyping system (Infinium genotyping assay (Illumina)), Affymetrix's Targeted Genotyping System for drug metabolizing enzymes and transports (DMETs) analysis (Affymetrix), and other mutations (such as SNPs) detection platforms, which are on the basis of the principles of nucleic acid hybridization-based reaction, single base extension reaction (Nikiforov et al., 1994; Bell et al., 2002), allele-specific primer extension and ligation reaction (Gunderson et al., 2005; Landegren et al., 1988), primer ligation reaction (Weiguo Cao, Clinical and Applied Immunology Reviews 2 2001:33-43), restriction enzyme reaction, padlock probe reaction (Xiaoquan Qi, Nucleic Acids Research, 2001, Vol. 29, No. 22 e116); and the relevant gene mutation detections (such as SNP detection) and nucleic acid sequencing analysis as described in other relevant patents, such as U.S. Pat. Nos. 5,427,930, 6,479,242, WO9300447, U.S. Pat. Nos. 5,871,921, 6,858,412, 5,866,337, etc.
Although those methods have some advantages per se, each also has some defects, such as for the chip technology based on direct nucleic acid hybridization, its non-specific hybridization may result in low resolution, and easily produce a false positive result; while for the chip technologies based on single base extension reaction and allele-specific primer extension reaction, both of them need a multi-color fluorescence system, as well as the amplification, preparation and purification of a target, so that large-scale mutation detections are time-consuming, labor-intensive, etc.
In view of the defects of the existing gene mutation detection (such as SNPs detection) and nucleic acid sequencing analysis, the inventor, on the basis of the multi-year accumulative practical experiences and expertise on the design and manufacture of such products and in combination with the application of related theories, actively does some researches and innovations, so as to create a new detection probe, a common oligonucleotide chip and a nucleic acid detection method, such that the general existing methods for gene mutation and DNA sequencing analysis are improved to be more practical. After continuous study, design, and repeated trial-manufacture and improvement of samples, the present invention, which indeed has a practical value, has been achieved finally.